This invention relates to the amplification of chromosomal DNA in situ. In particular, this invention relates to the amplification of chromosomal DNA in situ prior to microdissection.
Chromosome microdissection is an extremely useful molecular cytogenetic tool for the characterization and analysis of chromosomes. For example, microdissection has become a very popular method for making both whole-chromosome and region-specific painting probes for use in Fluorescence in situ Hybridization (FISH). In addition, microdissection has been applied to the isolation and characterization of region specific cDNAs.
While useful, microdissection does possess serious drawbacks, however. For instance, it is generally necessary to dissect multiple copies of a target chromosome or chromosome region to produce a probe sufficiently complex for FISH. Although band-specific probes have been made from single chromosome fragments, painting regions larger than this requires as many as 50 chromosome copies to be dissected for sufficient probe coverage. The need to dissect more than one copy of a target chromosome complicates the process of microdissection as it can be difficult to precisely locate the same chromosomal region when making multiple scrapes of a single band. As a result, the painting probe covers a wider region than desired. When probes are made for adjacent bands this can result in overlapping signals, which complicates analysis. Thus, the ability to make chromosome paints from single scrapes of a band, arm, or chromosome is highly desirable.
There is thus a need to amplify chromosomal DNA in situ in order to increase the amount of DNA associated with a chromosome or chromosome region to facilitate chromosome analysis by microdissection.
In order to meet these needs, the present invention is directed to the amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region. The chromosomes may be isolated from any chromosome-containing organism including birds (avian), reptiles, amphibians, plants, and mammals such as humans, mice and rats, etc. The chromosomes include mini chromosomes.
The amplification of chromosomal DNA in situ allows Fluorescence in situ Hybridization (FISH) painting probes to be made from single dissected chromosome fragments. Furthermore, the amplification of chromosomal DNA in situ permits the synthesis of cDNA libraries from low copy mRNAs. The amplification of chromosomal DNA in situ facilitates comparative hybridization and microdissection of tumor sections. In addition, the hybridization of cDNA libraries to chromosomes normalizes the frequency of the constituent cDNA sequences, i.e. increases the ratio of the less prevalent to more prevalent expressed sequences.
The present invention is directed to a method of preparing chromosomes for microdissection, comprising the steps of: a) fixing cells on a surface wherein the cells comprise chromosomes and the chromosomes include chromosomal DNA and b) amplifying the chromosomal DNA on the surface. The chromosomal DNA may be amplified by PCR including DOP-PCR.
The present invention is further directed to a method of microdissecting chromosomes, comprising the steps of: a) fixing cells on a surface wherein the cells comprise chromosomes and the chromosomes include DNA; b) amplifying the DNA in situ, and c) microdissecting the chromosomes. The DNA may be amplified by PCR including DOP-PCR in step b).
The present invention is further directed to a method of amplifying chromosomal DNA in situ, comprising the steps of: a) fixing cells on a surface wherein the cells comprise chromosomes and the chromosomes include DNA; b) preparing a PCR reaction buffer; c) adding the PCR reaction buffer to the fixed cells of step a); and d) amplifying the DNA by PCR in situ. The PCR amplification step d) may be DOP-PCR. The PCR reaction buffer will include DNA polymerase, eg. thermostable DNA polymerase.
The present invention is further directed to a method of amplifying chromosomal DNA in situ, comprising: a) providing cells embedded in paraffin wherein the cells comprise chromosomes and the chromosomes include DNA; b) preparing a PCR reaction buffer; c) combining the PCR reaction buffer with the paraffin embedded cells of step a) and d) amplifying the DNA by PCR in situ. The DNA in step d) may be amplified by DOP-PCR. This method may be used in comparative genomic hybridization (CGH) to compare the DNA content of cells, e.g. cancer cells vs. normal cells. For example, after DNA amplification in situ, CGH may be used on DNA isolated from tumor cells labeled with a fluorochrome of one color (e.g., green), and DNA from normal cells, usually but not necessarily obtained from a non-neoplastic region adjacent to the tumor, labeled with a different color (e.g., red). After hybridizing and washing steps, the chromosomes are analyzed for differential hybridization to compare and contrast cancerous and non-cancerous cells.
The present invention is further directed to a method of generating chromosome region-specific nucleic acids for a chromosomal region of interest comprising the steps of: a) obtaining chromosomes containing chromosomal DNA; b) amplifying the chromosomal DNA in situ, and c) microdissecting the chromosomal region of interest to provide a microdissected chromosome fragment containing microdissected chromosomal DNA. The amplification step b) may further comprise a polymerase chain reaction. The method may further comprise step e) labeling the amplified microdissected chromosomal DNA. The invention is further directed to labeled chromosomes and chromosome probes, and labeled chromosome region-specific nucleic acids for a chromosome region-specific nucleic acid produced by the method of the invention.
The DNA may be labeled using any conventional techniques, which permit detection. Examples of suitable labels include biotin-avidin immunofluorescent, digoxigenin, chromogenic, and radioisotopic labels and direct chemical labeling with fluorochromes.
The present invention is further directed to a method of localizing a chromosomal region of interest in a chromosome sample having nucleic acid sequences, comprising the steps of: a) providing a chromosome region-specific probe generated by: (i) amplifying the chromosomal DNA in situ, (ii) microdissecting the chromosomal region of interest to provide a microdissected chromosome fragment; (iii) amplifying the microdissected chromosome fragment; and (iv) labeling the amplified fragment to provide the probe; and b) contacting the chromosome sample with the probe under conditions favorable for hybridization between the probe and complementary nucleic acid sequences in the sample and c) determining the existence and location of hybridization in the chromosome sample.
The present invention is further directed to a method of screening a library of nucleic acid clones for a clone of a chromosomal region of interest comprising the steps of: a) providing a chromosome region-specific probe generated by: (i) amplifying the chromosomal DNA in situ, (ii) microdissecting the chromosomal region of interest to provide a microdissected chromosome fragment; (iii) amplifying the microdissected chromosome fragment; and (iv) labeling the amplified fragment to provide the probe; b) providing the library of clones to be screened; c) contacting each clone with the probe under conditions favorable for nucleic acid hybridization; and d) determining whether and in which clone hybridization has occurred.
The present invention is further directed to a method of amplifying a cDNA library, comprising: a) fixing cells on a surface wherein the cells comprise chromosomes and the chromosomes include genomic DNA; b) amplifying the genomic DNA on the surface to form amplified genomic DNA; c) hybridizing the cDNA library to the genomic DNA to form DNA hybrids; and d) amplifying the DNA hybrids on the surface to form an amplified cDNA library. In the method, the DNA in steps b) and d) may be amplified by PCR; e.g. DOP-PCR. The method may also include the additional step e) microdissecting the chromosomes and/or step f) amplifying the DNA hybrid from the microdissected chromosomes.
The present invention is further directed to a method of preparing a cDNA library specific to a particular chromosome region, comprising: a) fixing chromosomes including a chromosome region on a surface wherein the chromosomes include genomic DNA; b) amplifying the genomic DNA in situ to form amplified genomic DNA; c) hybridizing the cDNA library to the genomic DNA to form DNA hybrids; d) amplifying the DNA hybrids on the surface to form an amplified cDNA library; e) microdissecting the chromosome region wherein the chromosome region includes a portion of the amplified cDNA library; and f) amplifying the portion of the cDNA library. In the method, the amplification steps d) and f) may be with PCR primers specific for the cDNA library.
The present invention is further directed to a method of identifying expressed genes, comprising: a) fixing cells on a surface wherein the cells comprise chromosomes and the chromosomes include genomic DNA; b) amplifying the genomic DNA on the surface to form amplified genomic DNA; c) hybridizing the cDNA library to the genomic DNA to form DNA hybrids; d) amplifying the DNA hybrids on the surface to form an amplified cDNA library; and e) sequencing members of the amplified cDNA library to identify the expressed genes. In the method, the amplifications of steps b) and d) may be by PCR with primers specific for the cDNA library.
This invention is further directed to method of normalizing a cDNA library, comprising: a) fixing cells on a surface wherein the cells comprise chromosomes and the chromosomes include genomic DNA; b) amplifying said genomic DNA on said surface to form amplified genomic DNA; c) hybridizing the cDNA library to the amplified genomic DNA to form DNA hybrids; d) amplifying the DNA hybrids on the surface to form an amplified cDNA library; e) microdissecting the chromosomes including the amplified cDNA library; and f) amplifying the amplified cDNA library to form a normalized cDNA library.